Current methods for separating biopolymer conjugated molecules (e.g., PEGylated molecules) from unconjugated molecules (e.g., unPEGylated molecules) require many steps. For example, such methods include the following steps: 1) preparative purification of the unconjugated molecule by ion-exchange high performance liquid chromatography (HPLC) under gradient conditions, with collection of fractions, 2) analysis of the fractions by analytical HPLC, 3) pooling of the fractions and analytical HPLC analysis of the appropriate fraction pools, 4) desalting of the appropriate fraction pool, 5) lyophilization of the unconjugated molecule, 6) conjugating a biopolymer to the molecule (e.g., PEGylating the molecule), 7) application of the crude biopolymer (e.g., PEG) reaction mixture to another ion-exchange HPLC under gradient conditions, with collection of the fractions, 8) analyzing the fractions by analytical HPLC, 9) pooling of the fractions and analysis by analytical HPLC, 10) performing ultrafiltration and desalting, and 11) lyophilizing the biopolymer conjugated molecule (e.g., PEGylated molecule). Every purification will decrease the total yield of the desired product because some fractions will inevitably contain a mixture of products. In addition, the analysis of the individual fractions and fraction pools increases time spent in a manufacturing plant, as well as the cost of the analysis itself.
For large scale (e.g., GMP (good manufacturing practices)) manufacturing of biopolymer conjugated molecules, streamlining the aforementioned protocol would result in a significant reduction in both production cost and time. Those skilled in the art of process chemistry and engineering will recognize the need to eliminate purifications and reduce the number of overall unit operations. Accordingly, there is a need for the development of quick and efficient processes for the purification in high yield and purity of biopolymer conjugated molecules, such as, for example, oligonucleotides, such as aptamers, and proteins that are conjugated to polyethylene glycol moieties.